Means and methods for the microbial production of numerous polypeptides, including human growth hormone, were disclosed by Goeddel et al. in U.S. Ser. No. 55,126, filed July 5, 1979, which is hereby incorporated by reference. Counterparts of the application have been published, e.g. British patent application publication No. 2,055,382 and European patent application publication No. 22242.
Methods for producing various heterologous polypeptides from microorganism hosts have required the identification of the DNA sequence encoding the particular desired product. Once known, the sequence could be fashioned, synthetically or from tissue derived cDNA, and operably inserted into expression vectors that were then used to transform the host organism and thus direct its production of the polypeptide. In the past, relatively small proteins could be produced by synthesizing the entire gene. The first example of this was human insulin (see British patent application publication No. 2,007,676, for example). For polypeptides too large to admit of microbial expression from entirely synthetic genes, cDNA was obtained from mRNA transcripts derived from tissue.
In each case, knowledge of the sequence of the amino acid was essential so that the correct sequential synthesis was conducted in the one case and that correct homologous sequence probes for isolating appropriate mRNA sequences could be used in the other.
Problems are encountered where the sequence of the polypeptide is unknown and the source of the polypeptide contains insufficient amounts to insure extraction of enough material for sequencing. Thus, hitherto employed methods fail where the gene is expressed in unknown tissue or in undetectable amounts or in very few cells of a tissue or because of other reasons related to limitations imposed by the current state of the art concerning the isolation of rare mRNA.
It was perceived that improved refinements in recombinant DNA techniques would be useful in providing desired heterologous protein under such circumstances.